Pyrrolo-quinoline quinone (PQQ) is a non-covalently bound prosthetic group of a class of bacterial dehydrogenases that catalyze the oxidation of alcohols and sugars in the periplasm of Gram-negative bacteria. PQQ has also been shown to be necessary for proper growth and development in mice, and recent evidence has been presented that suggests PQQ may be an essential B vitamin in mammals. The role of PQQ as a prokaryotic redox co-factor has received significant attention, but the biogenesis of PQQ is much less understood. This research proposal will investigate the primary steps in the mechanism for PQQ biogenesis by using in vitro studies of PqqE to isolate and characterize the intermediates and products of this reaction. Protein homology studies have indicated that PqqE is part of a class of proteins known as Radical SAM (S- adenosylmethionine) enzymes, that contain a [4Fe-4S] cluster in the active site. These enzymes catalyze unique reactions via the formation of an organic radical from reductive activation of SAM by the iron sulfur cluster. Recombinant expression systems will be used to express PqqE, and in vitro studies will be initiated to understand the specific biochemical intermediates, rate constants, and products. Solid phase peptide synthesis will be invoked to synthesize the peptide substrate (PqqA) for PqqE, along with creating structural analogs that will be used to trap the intermediates proposed in the reaction mechanism. HPLC and LC-MS will be used in conjunction with various radical scavenging agents to isolate and characterize the products of the anaerobic and aerobic reactions. Steady state and freeze-quench EPR studies will provide an added tool for understanding the radical intermediates formed during the PqqE reaction. These will be done in conjunction with substrates specifically labeled with deuterium and carbon-13 to make definitive assignments of the radical intermediates formed during the reaction mechanism. The long term goal of this project is to elucidate the biochemical steps and mechanisms for PQQ production. These studies will have important implications for human health because they will provide insight into a biochemical pathway specific for gram-negative bacteria, which may yield added tools for a new class of antibiotic targets. Understanding the biochemical mechanisms in the PqqE reaction may also yield important information concerning other enzymes of the Radical SAM super family, that have been implicated in a variety of different reactions in humans. Specifically, one question we would like to address is, whether PqqE can function as a putative Radical SAM enzyme and an oxygenase. [unreadable] [unreadable] [unreadable]